Deposition of aggregates of misfolded protein into intracellular inclusion bodies (IB) is a prominent cytopathological feature of nearly every known neurodegenerative disease. A confluence of recent data has challenged the widespread belief that IB are pathogenic. Studies from conditional animal models of several neurodegenerative diseases including tauopathy, Huntington's disease and spinocerebellar ataxia type 7 reveals that neurons are endowed with the capacity to recover from the toxicity of misfolded mutant proteins, and are able to clear intracellular IB. Together, these findings support a cytoprotective role for IB formation. We have demonstrated that dynein-dependent transport of polyubiquitinated misfolded or aggregated proteins are delivered to cytoplasmic IB via dynein-dependent transport on microtubule tracks. Such dynein-dependent IB are called aggresomes (AG). We previously hypothesized that AG formation can contribute to clearance of toxic misfolded or aggregated proteins by facilitating their degradation in lysosomes by autophagy, and recent studies strongly support a role for autophagy as a cytoprotective mechanism in human disease and animal models thereof. The studies proposed here are focused on elucidating the mechanisms by which potential proteotoxins are recognized and transported to AG for autophagic degradation. To this end, three specific aims are proposed. The first aim will use novel synthetic substrates to assess the roles of polyubiquitination and aggregation as cis-acting signals for targeting to AG in vivo and in cell-free extracts. The second aim will exploit a novel cell-free AG formation assay to purify and identify trans-acting factors that couple AG substrates to cytoplasmic dynein. The third aim will define the structural and functional relationship between AG formation and autophagic degradation of misfolded or aggregated proteins using a novel assay and genetic screen in yeast. PUBLIC HEALTH REVELANCE: The formation of toxic protein aggregates underlies the pathogenesis of most neurodegenerative disorders. Cells are known to possess mechanisms to counteract the formation of and to eliminate these toxic protein species, but the mechanisms by which this occurs is not understood in sufficient detail to develop viable therapeutic strategies. The research in this proposal will use biochemical and genetic means to understand the mechanism by which cells can protect themselves against toxic aggregated proteins.